See the article "The 1918–1919 pandemic of
H1N1 virus influenza was the greatest acute plague of the 20th century.
Incurring over 20 million human fatalities, however, was not a good
strategy for sustaining the evolutionary fitness of the virus, because
it is no longer extant; whereas, say, measles and chickenpox remain
with us with no evidence of remarkable genetic change, although this
may become more evident if they were to face total or near eradication
through vaccination programs. The folly of flu virulence remains our
chagrin, because the threat always looms over us that this family of
viruses, endemic in birds, again may generate human-lethal gene
reassortments. We had valid scares about that contingency with the
appearance of H5N1 variant flu in Hong Kong just 3 years ago. Influenza
can be regarded as a zoonosis prevalent in birds, many of them world
travelers, with occasional outbreaks in humans and other animals mainly
rooted in nature's own experiments in genetic engineering. Special
importance is attached to reassortments between bird- and human-adapted
strains most likely to occur in habitats with close contact between
birds, e.g., ducks, humans, and swine (as a mixing reservoir; ref. 1).
For these reasons, high urgency attaches to efforts to resurrect
genetic information about the singularities of H1N1–1918. The intact
virus is nowhere to be found, but genomic fragments can still be
detected sensitively and diagnosed. Exemplifying the latest technical
advances in the use of DNA amplification, reverse-transcriptase–PCR
(RT-PCR), Jeffery Taubenberger and his associates at the Armed Forces
Institute of Pathology initiated the tour de force of recovering
sequences of flu from paraffin-embedded pathological specimens
preserved since 1918 in the AFIP collections (2). These sources then
were augmented by samples from frozen remains of an Inuit woman who
succumbed to the flu in 1918 and was buried in permafrost at Brevig
Mission on the Seward Peninsula of Alaska's western coast, not far
from the Bering Strait. This nameless woman has left an indelible mark
on world medical history (3). Now, as reported in this issue, the AFIP
team has joined forces with teams from the U.S. Department of
Agriculture and the Peter Palese/Adolfo García-Sastre groups
at Mt. Sinai Medical School in a further quest for the RNA sequences of
H1N1–1918 that might account for its historic human virulence (4).
The flu genome comprises about 13,500 bases of single-stranded RNA,
disposed in eight segments varying from approximately 900 to 2,341
each. This genome is only a few millionths of the complexity of the
human genome, but it is organized with great efficiency, lacks “junk
R/DNA,” and encodes for a short dozen of identified gene products
(Fig. (Fig.1).1
). Many strains of flu have been
sequenced fully; this feat will be achieved for H1N1–1918 with arduous
labor, because the RNA, although frozen, is fragmented into snippets no
larger than approximately 120 bases each. The practical way now
available is to devise probes by using segments from extant flu
strains, guessing at possible homologous strings, or synthesizing
probes with calculated degeneracy. Until a complete genomic sequence is
achieved, and it is hard to see how that will be authenticated, it is
possible even that H1N1–1918 contains extraneous inserted sequences
quite foreign to the canonical flu strains. Very reasonably, initial
efforts focus on flu genes already identified in viruses recovered from
recent outbreaks in humans, birds, swine, and other animals.
). Many strains of flu have been
sequenced fully; this feat will be achieved for H1N1–1918 with arduous
labor, because the RNA, although frozen, is fragmented into snippets no
larger than approximately 120 bases each. The practical way now
available is to devise probes by using segments from extant flu
strains, guessing at possible homologous strings, or synthesizing
probes with calculated degeneracy. Until a complete genomic sequence is
achieved, and it is hard to see how that will be authenticated, it is
possible even that H1N1–1918 contains extraneous inserted sequences
quite foreign to the canonical flu strains. Very reasonably, initial
efforts focus on flu genes already identified in viruses recovered from
recent outbreaks in humans, birds, swine, and other animals.
| Figure 1 Diagram of an influenza-virus particle. The surface of each influenza
virion consists of a lipid envelope in which two major viral surface
antigens, the hemagglutinin (HA) and the neuraminidase (NA), are found.
Within the particle are the eight negative-sense (more ...) |
Previous work has focused on two well studied gene products:
hemagglutinin (HA) and neuraminidase (NA), which dominate the surface
specificities of the virus and underlie most of its taxonomy (e.g.,
H1N1 refers to type 1 hemagglutinin, type 1 neuraminidase). These gene
products are also the chief determinants of specificity in vaccine
prophylaxis for flu strains circulating at any given time. HA variation
can account for fluctuations of virulence and host specificity of
extant flu viruses. However, nothing remarkable was seen in the HA or
the NA of H1N1–1918. The next gene to be scrutinized now is NS1
(nonstructural protein 1), which the Palese/García-Sastre
groups have fingered recently as an interferon antagonist and as gene
essential for flu virulence in a mouse model. A reasonable conjecture
was that the hypervirulence of H1N1–1918 might be lodged in its NS1,
and this might be revealed in reinsertions of the 1918-NS1 segment into
mouse-adapted flu strains. This challenging construct was generated in
the laboratory—one hastens to footnote, under BL-3+ conditions, and
under the USDA's stern regulatory scrutiny—and tested in mice. The
unexpected and perhaps disappointing result was the mitigation not
enhancement of virulence in this species. The incapacitation of the
NS1-virulence function in the mouse was ascribed to interaction with
its host factors; the other variable would be other elements of the
genome of the mouse-adapted flu strain. NS1 singularity for the
human virulence of H1N1–1918 is neither falsified nor
corroborated by these findings.
There still remain a handful of gene candidates, including the
polymerases essential for the replication of the virus. This label does
not preclude any of them from also functioning in networks and pathways
that are expressed as virulence. It should caution us about the
nominalist fallacy to recall that the δ crystallin of the bird's
lens does double duty as argininosuccinate lyase, an enzyme in the urea
cycle.
In principle, the NS1 hypothesis (and its alternatives) might be tested
by using similar gene constructs based on flu viruses adapted to other
animal species, including primates, and challenging the corresponding
hosts. Negative results would be as inconclusive as those with the
mouse. Positive results, namely the association of hypervirulence with
a gene sequence borrowed from N1H1–1918, would be a great advance in
medical science and would offer constructive models for the development
of prophylactic and therapeutic measures. They would also induce great
alarm about the potential hazards to human health, if humans were also
susceptible, and the virus might escape. Any such experiments should be
done with strains for which current vaccines are disseminated
widely and have proven effectiveness.
To conduct such experiments with human-adapted strains and challenge to
human subjects as the probative step, is well nigh unthinkable. But
nature is under no such restraint! The current results are a caution to
look closely at the involvement of NS1 (as well as HA and NA) variation
in natural outbreaks in many species and to look out for their
reassortment into human strains. In addition, it might be well to
undertake a special search for close homologues to 1918-NS1 in viruses
circulating in avian and other species, in which they may appear to be
benign in their current hosts (as in the present mouse experiments).
That would be nature's inverse of the current report.
The publication by Basler et al. (4) will attract great
admiration for its technical finesse and will serve as an example of
the fruits from convergence of natural history, field exploration,
clinical insight, and sophisticated molecular wizardry. It also will
awaken anxieties about the obvious opportunities for abuse. The really
fateful step was taken with the very first cultivation of pathogenic
bacteria and viruses a century ago—perhaps most importantly with the
discovery of the concepts of germs and communicable diseases. The
notion of using ever more sophisticated technology for intentionally
constructing or reconstructing ever more pathogenic variants lends
further weight to that anxiety. The great debate of the mid-1970s led
to sensible measures for the regulation of recombinant DNA research.
There has been increasing understanding that some of nature's
pathogens deserve equal or greater respect. We should be sure that we
continue to devote as much reasoned ingenuity to the design of
safeguards and to informed and transparent third-party scrutiny of
potential hazards as we do generally to the authentication of
scientific claims. We cannot afford to forego the deepest research into
the plagues that beset humankind. Nor can we afford to blunder into
mistakes that will do primary injury to bystanders and incur
incommensurate social sanctions.
My deepest anxieties pertain to the smoldering technology and arms race
that attends the power struggles in the Middle East and the economic
instabilities of the former Soviet Union. Although the 1975 Biological
Weapons Convention (BWC) has demilitarized the main drivers of
bioweaponry technical advance, in the U.S. and in the overt activities
of other formidable powers, the BWC has not been enforced successfully
against Iraq and is more or less openly flouted in a handful of other
countries. The United Nations (UN) Security Council is too splintered
on other issues to take a firm stand on the defiance by Iraq of the
UN-mandated inspections. It would not be child's play for defiant
small countries to adopt advanced biotechnology into their weapons
programs. But we have seen that the climactic high-science successes in
one decade become fodder for high-school projects in the next.
Influenza is an unlikely candidate for rational weapons development,
because new strains promptly embrace the world. But that logic is
insufficient reason to neglect the contingency. More likely similar
principles would be applied to more governable bioagents, but any
bioagents in warfare are an affront and a threat to the entire human
species. Informed professionals throughout the world should be leading
campaigns to insist on universal compliance with the BWC as a major
bulwark of human health and associating that with the most positive
measures to apply advanced biotechnology in a constructive way for
dealing with nature's continued scourges.